Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
  • G01N33/533—Production of labelled immunochemicals with fluorescent label
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/81—Packaged device or kit
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/80—Fluorescent dyes, e.g. rhodamine
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/805—Optical property
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
  • Y10S436/808—Automated or kit
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/815—Test for named compound or class of compounds
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/815—Test for named compound or class of compounds
  • Y10S436/817—Steroids or hormones

Definitions

• This invention relates to assay methods, and reagent means for use therein, of the homogeneous and heterogeneous specific binding type for determining qualitatively or quantitatively a ligand in, or the ligand binding capacity of, a liquid medium.
• the invention relates to an improved fluorescer-labeled specific binding assay.
• compositions include a labeled conjugate comprising a binding component incorporated with a label, which labeled conjugate participates with other constituents, if any, of the reagent means and the ligand or ligand binding capacity in the medium under assay to form a binding reaction system producing two species or forms of the labeled conjugate, a bound-species and a free-species.
• the relative amount or proportion of the labeled conjugate that results in the bound-species compared to the free-species is a function of the presence (or amount) of the ligand or ligand binding capacity to be detected in the test sample.
• the reagent means would comprise (1) a labeled conjugate in the form of the ligand to be detected (e.g., an antigen or hapten) chemically linked to a label, and (2) a specific binding partner for the ligand (e.g., an antibody).
• a labeled conjugate in the form of the ligand to be detected e.g., an antigen or hapten
• a specific binding partner for the ligand e.g., an antibody
• the amount of labeled conjugate that becomes bound to the binding partner i.e., that which results in the bound-species
• that amount which remains free i.e., unbound to the binding partner and thus that which results in the free-species
• the amount of labeled conjugate resulting in either species is determined by measuring, i.e., monitoring, the label therein.
• the bound-species and the free-species must be physically separated in order to complete the assay.
• This type of assay is referred to in the art as "heterogeneous".
• a "homogeneous" format can be followed and the separation step avoided.
• the first discovered type of highly sensitive specific binding assay was the radioimmunoassay which employs a radioactive isotope as the label. Such an assay necessarily must follow the heterogeneous format since the monitorable character of the label is qualitatively unchanged in the free- and bound-species. Because of the inconvenience and difficulty of handling radioactive materials, many new assay systems have been devised using materials other than radioisotopes as the label component, including enzymes, bacteriophages, metals and organometallic complexes, coenzymes, enzyme substrates, enzyme inhibitors, cyclic reactants, organic prosthetic groups, chemiluminescent reactants, and fluorescent molecules.
• Certain fluorescent binding assays of the homogeneous type have been conceived whereby the usually disadvantageous separation step can be avoided.
• One such method the fluorescence polarization technique, is described in U.S. Pat. No. 4,115,699 and is based on the observation that irradiation with polarized light of certain fluorescer-ligand conjugates when bound by a binding partner (e.g., antibody) results in emission of light which has a different polarization.
• a binding partner e.g., antibody
• Another homogeneous fluorescent binding assay is based on quenching or enhancement of fluorescence upon binding of a fluorescer-ligand conjugate by its binding partner. Examples of these techniques are provided by the descriptions in Belgian Pat. No. 858,722 and German Offenlegungsschriften Nos. 2,716,276 and 2,716,515. A variation of this assay method is described in U.S. Pat. No. 3,996,345 which employs a specific quenching substance as a counterpart to the fluorescer label.
• Chemiluminescent binding assays are the subject of U.S. Pat. No. 4,104,029 and U.S. patent applications Ser. Nos. 894,836 and 894,838, filed Apr. 10, 1978, which applications are assigned to the present assignee.
• the chemiluminescent-based assays specifically described employ consumable reactants as the label, thereby theoretically limiting sensitivity, and, more significantly, are susceptible to protein quenching.
• the present invention provides an improvement in a specific binding assay method for determining a ligand in, or the ligand binding capacity of, a liquid medium, wherein the liquid medium is combined with reagent means including a labeled conjugate comprising a binding component incorporated with a fluorescent label, which combination forms a binding reaction system having a bound-species and a free-species of the labeled conjugate, the amount of said fluorescent label resulting in either of the bound or free-species being a function of the presence or amount of the ligand or ligand binding capacity in the liquid medium, and wherein said label is measured in one or both of the species of the labeled conjugate.
• the improvement comprises accomplishing the measurement step by chemically exciting the label to cause the same to emit light, and then measuring the light emitted by the excited fluorescer.
• the fluorescent label is preferably excited by exposure to a substance capable of causing such excitation and light emission, for example by exposure to a high energy intermediate reaction product produced by reaction, in the presence of said label, of hydrogen peroxide and oxalyl chloride, an oxamide, or a bis-oxalate ester.
• the present assay method may follow any conventional homogeneous or heterogeneous assay technique.
• the improved reagent means comprises the reagents appropriate for the binding reaction system which is used and, in combination therewith, the chemical means for producing the high energy substance capable of exciting the fluorescer label, i.e., the plurality of reagents which react to produce the high energy intermediate reaction product.
• such means include (i) hydrogen peroxide or a conventional chemical system for generating hydrogen peroxide and (ii) oxalyl chloride, an oxamide, or a bis-oxalate ester.
• the monitoring reaction is accomplished in the desired species of the binding reaction system (i.e., in the separated bound or free-species when following a heterogeneous format or in the combined species when following a homogeneous format) by addition of the reagents which chemically react to produce a high energy reaction product capable of transferring energy to the fluorescer label to raise same to an excited state.
• the excitation of the fluorescer decreases toward a ground state of energy, energy is emitted in the form of light.
• hydrogen peroxide and various reactive bis-oxalate esters react to form a high energy intermediate believed to be either 1,2-dioxethanedione or activated carbon dioxide.
• the present invention has several advantages over the prior art fluorescent and chemiluminescent binding assays.
• One advantage is that relatively simple detection instrumentation may be used, and consequently the necessities of a light source, band filters, and allowances or corrections for scattering of the incident light to the photodetector, all required in fluorometric measurements, are obviated.
• a further advantage over standard fluorometric detection is that because no incident light is required to initiate light production, all of the light produced, without wavelength restrictions, can be measured.
• a theoretical advantage over the prior art chemiluminescent binding assays is that of improved sensitivity by reason of the fact that the label does not behave as a consumable reactant.
• the present assay is generally free of problematic protein quenching found in prior art chemiluminescent assays.
• ligand is the substance, or class of related substances, whose presence or the amount thereof in a liquid medium is to be determined;
• specific binding partner of the ligand is any substance, or class of substances, which has a specific binding affinity for the ligand to the exclusion of other substances;
• specific binding analog of the ligand is any substance, or class of substances, which behaves essentially the same as the ligand with respect to the binding affinity of the specific binding partner for the ligand;
• reagent means is a composition, device or test kit comprising the reagents used to perform the present assay method.
• the present assay may be applied to the detection of any ligand for which there is a specific binding partner and, conversely, to the detection of the capacity of a liquid medium to bind a ligand (usually due to the presence of a binding partner for the ligand in the medium).
• the ligand usually is a peptide, protein, carbohydrate, glycoprotein, steroid, or other organic molecule for which a specific binding partner exists in biological systems or can be synthesized.
• the ligand in functional terms, is usually selected from the group consisting of antigens and antibodies thereto; haptens and antibodies thereto; and hormones, vitamins, metabolites and pharmacological agents, and their receptors and binding substances.
• ligands are immunologically-active polypeptides and proteins of molecular weights between 1,000 and 4,000,000, such as antibodies and antigenic polypeptides and proteins, as well as haptens of molecular weights between 100 and 1,500.
• antigenic polypeptides are angiotensin I and II, C-peptide, oxytocin, vasopressin, neurophysin, gastrin, secretin, and glucagon.
• antigenic proteins are insulin, chorionic gonadotropin (e.g., HCG), carcinoembryonic antigen (CEA), myoglobin, hemoglobin, follicle stimulating hormone, human growth hormone, thyroid stimulating hormone (TSH), human placental lactogen, thyroxine binding globulin (TBG), intrinsic factor, transcobalamin, enzymes such as alkaline phosphatase and lactic dehydrogenase, and hepatitis-associated antigens such as hepatitis B surface antigen (HB s Ag), hepatitis B e antigen (HB e Ag) and hepatitis B core antigen (HB c Ag).
• HCG chorionic gonadotropin
• CEA carcinoembryonic antigen
• myoglobin hemoglobin
• follicle stimulating hormone human growth hormone
• TSH thyroid stimulating hormone
• TSG human placental lactogen
• TMG thyroxine binding globulin
• TMG
• antibody ligands are those antibodies of the IgG, IgE, IgM and IgA classes specific for any of the antigens or haptens herein described.
• the class of hapten ligands are exemplified by thyroxine, liothyronine, the estrogens such as estriol, prostaglandins, vitamins such as biotin, vitamin B 12 , folic acid, vitamin E, vitamin A, and ascorbic acid (vitamin C), and drugs such as carbamazepine, quinidine, digoxin, digitoxin, theophylline, phenobarbital, primidone, diphenylhydantoin, morphine, nicotine, and so forth.
• the liquid medium to be assayed can be a naturally occurring or artificially formed liquid suspected of containing the ligand or a binding capactiy therefor, and usually is a biological fluid or a liquid resulting from a dilution or other treatment thereof.
• Biological fluids which can be assayed following the present method include serum, plasma, urine, saliva, and amniotic and cerebrospinal fluids.
• Other materials such as solid matter, for example tissue, can be assayed by reducing them to a liquid form, such as by dissolution of the solid in a liquid or by liquid extraction of the solid.
• the fluorescer used according to the present invention may be any fluorescent substance, that is, any substance which, upon excitation to a high energy state, such as by illumination or upon exposure to a high energy intermediate reaction product in accordance with the present invention, thereafter radiates or emits light.
• the phenomenon of fluorescence is the result of changes in the energy state of electrons in the electronic configuration of the fluorescer.
• electrons are excited to a high energy state and, upon decay of that energy state to a lower, more stable level, energy is emitted in the form of light. Because of attendent energy loss in forms other than light during the fluorescence, the emitted light is generally of a lower energy and, accordingly, of a longer wavelength than the incident light.
• fluorescers particularly preferred are those having an organic structure which is readily suited to synthetic coupling to a binding component in the particular binding reaction system to be employed so as to form the necessary labeled conjugate.
• preferred fluorescers are lissamine rhodamine B, rhodamine B, fluorescein, 9,10-diphenylanthracene, perylene, rubrene, pyrene, or fluorescent derivatives thereof, such as the isocyanate, isothiocyanate, acid chloride, or sulfonyl chloride derivatives.
• the fluorescer is coupled to the selected binding component for the assay (such component will usually be the ligand, a specific binding analog of the ligand, or a specific binding partner of the ligand as will be described in detail below) directly or, more usually, through a bridge group such that the desired fluorescent property of the fluorescer-label is retained in the labeled conjugate.
• a bridge group such that the desired fluorescent property of the fluorescer-label is retained in the labeled conjugate.
• such bridging group usually comprises between 1 and 50, more usually between 1 and 15, carbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur, and phosphorus) in the chain.
• the bridging group usually has a molecular weight not exceeding 1000 and usually less than 200.
• the precise chemical structure of the bridging group, the linkages between the fluorescer-label and the binding component, and the respective terminal groups of the bridging group will all depend upon the precise nature of the fluorescer and binding component involved.
• the synthetic coupling process which forms the fluorescer-labeled binding component i.e., the labeled conjugate is well within the skill of the art.
• oxamides such as 1,1'-oxalyl-bis-(benzimidazole), N,N'-dimethyl-N,N'-dinitrooxamide, and N,N'-bis-(phenylsulfonyl) parabamate
• bis-oxalate esters such as bis-(2,4-dinitrophenyl) oxalate, bis-(pentachloro-phenyl) oxalate, bis-(4-nitro-3-trifluoromethylphenyl) oxalate, bis-(4-nitro-2-formylphenyl) oxalate and bis-(pentafluorophenyl) oxalate.
• the present assay can follow any of the conventional homogeneous or heterogeneous assay techniques. Following are brief summaries of various types of homogeneous and heterogeneous assay techniques that are available.
• a homogeneous assay technique i.e., an assay technique which does not require a physical separation of the bound-species and the free-species, is available where reaction between the binding component in the labeled conjugate and a corresponding binding partner causes a measurable change, either in a positive or a negative sense, in the light emitted by the fluorescer-label upon chemical excitation as described herein.
• the distribution of the fluorescer-label between the bound-species and the free-species can be determined by adding the reagents necessary to form the high energy intermediate directly to the combined species and measuring the light emitted (usually in terms of total light produced or peak intensity).
• Several manipulative techniques are available for carrying out a homogeneous assay with the two most common techniques being the direct binding and the competitive binding techniques.
• a liquid medium suspected of containing the ligand to be detected is contacted with a conjugate comprising the fluorescer-label coupled to a specific binding partner of the ligand, and any change in light emission by the fluorescer-label is assessed.
• the liquid medium is contacted with a specific binding partner of the ligand and with a labeled conjugate comprising the fluorescer-label coupled to the ligand or a specific binding analog thereof, and thereafter any change in light emission by the fluorescer-label is assessed.
• the fluorescer-label is measured by contacting the liquid medium with the reagents which react to produce the high energy intermediate capable of exciting the label.
• Qualitative determination of the ligand in the liquid medium involves comparing the light emission from assay of the liquid medium to that in liquid media containing known amounts of the ligand.
• the components of the specific binding reaction i.e., the liquid medium suspected of containing the ligand, the labeled conjugate, and/or a specific binding partner of the ligand, may be combined in any amount, manner, and sequence, provided that the light emission of the fluorescer-label is measurably altered when the liquid medium contains the ligand in an amount or concentration of significance to the purposes of the assay.
• all of the components of the specific binding reaction are soluble in the liquid medium.
• the components of the binding reaction are the liquid medium suspected of containing the ligand and a quantity of a conjugate comprising the fluorescer-label coupled to a specific binding partner of the ligand.
• the light emission of the conjugated fluorescer on contact with the liquid medium varies (usually inversely) with the extent of binding between the ligand in the liquid medium and the specific binding partner in the labeled conjugate.
• the light emission from the conjugated fluorescer will usually decrease.
• the components of the binding reaction are the liquid medium suspected of containing the ligand, a quantity of a conjugate comprising the fluorescer-label coupled to the ligand or a specific binding analog of the ligand, and a quantity of a specific binding partner of the ligand.
• the specific binding partner is contacted simultaneously or sequentially with the labeled conjugate and the liquid medium. Since any ligand in the liquid medium competes with the ligand or specific binding analog thereof in the labeled conjugate for binding with the specific binding partner, the light emission of the conjugated fluorescer on contact with the liquid medium varies (usually directly) with the extent of binding between the ligand in the liquid medium and the specific binding partner. Thus, as the amount of the ligand in the liquid medium increases, the light emission from the conjugated fluorescer will usually increase.
• Determination of the ligand binding capacity of (e.g., the presence of a specific binding partner of the ligand in) a liquid medium can be accomplished by a homogeneous technique by contacting the liquid medium with a conjugate comprising the fluorescer-label coupled to the ligand or a specific binding analog thereof.
• the light emission of the conjugated fluorescer on contact with the liquid medium varies (usually inversely) with the extent of binding between the binding capacity of the liquid medium and the ligand or analog thereof in the labeled conjugate similarly to the binding in a direct binding technique for determining the ligand as described above.
• the use of a fluorescer-label can also be applied to the conventional heterogeneous type assays wherein the bound- and free-species of the labeled conjugate are separated and the quantity of label in one or the other is determined.
• the reagent means for performing such a heterogeneous assay may take on many different forms. In general, such means comprises two basic constituents, which are (1) a specific binding partner of the ligand, and (2) a labeled conjugate which is normally a labeled form of (a) the ligand, (b) a specific binding analog of the ligand, or (c) the specific binding partner.
• the binding reaction constituents are combined simultaneously or in a series of additions with the liquid medium to be assayed and with an appropriate incubation period or periods, the labeled conjugate becomes bound to its corresponding competing binding partners such that the extent of binding, i.e., the ratio of the amount of labeled conjugate bound to a binding partner (the bound-species) to that unbound (the free-species), is a function of the amount of ligand present.
• the extent of binding i.e., the ratio of the amount of labeled conjugate bound to a binding partner (the bound-species) to that unbound (the free-species)
• insolubilizing agents are specific precipitating antibodies, specific insolubilized antibodies, and, where B or L is a proteinaceous material, protein precipitators such as ammonium sulfate, or where B or L is a small adsorbable molecule, dextran-coated charcoal. Description of parallel systems can be found in Biochem. J. 88:137 (1973) and U.S. Pat. No. 3,839,153.
• the reagent means of the present invention comprises all of the essential chemical elements required to conduct a desired assay method encompassed by the present invention.
• the reagent means is presented in a commercially packaged form, as a composition or admixture where the compatability of the reagents will allow, in a test device configuration, or as a test kit, i.e., a packaged combination of containers holding the necessary reagents.
• Included in the reagent means are the reagents appropriate for the binding reaction system desired, always requiring a labeled conjugate as defined hereinbefore.
• binding reaction reagents can include, in addition to the labeled conjugate, a binding partner to the ligand and so forth. Where a heterogeneous binding format is desired, the binding reaction reagents can include an insolubilized reagent or chemical means for rendering either of the bound- or free-species insolubilized.
• a typical set of binding reaction reagents for performing a homogeneous competitive binding assay for an antigen ligand would be (1) a fluorescer-labeled form of the ligand or a binding analog thereof and (2) an antibody to the antigen.
• a typical heterogeneous competitive binding assay set of reagents for an antigen ligand might be (1) a fluorescer-labeled form of the ligand or a binding analog thereof and (2) an insolubilized form of an antibody to the antigen.
• the present reagent means also includes one of the previously described plurality of reagents which react in the presence of the fluorescent label to produce a high energy intermediate reaction product which thereupon excites the label to a high energy state and causes it to emit light.
• such plurality of reagents comprises (i) hydrogen peroxide or any conventional chemical system generating hydrogen peroxide and (ii) oxalyl chloride, an oxamide, or a bis-oxalate ester, the latter two classes of compounds including the preferred species described previously herein.
• the reagent means can include other reagents as are known in the art and which may be desirable from a commercial and user standpoint, such as buffers, diluents, standards, and so forth.
• the reagent means of the present invention can comprise any elements known in the art of the heretofore available specific binding assay reagent means wherein the label is the fluorescer-label of the present invention and such reagent means additionally include one of the above described plurality of reagents for generating the high energy intermediate reaction product.
• Lissamine rhodamine B was obtained from Pfaltz and Bauer (Stamford, Connecticut, USA), hydrogen peroxide (50%) from Fischer Scientific (Fairlawn, New Jersey, USA), and sisomicin from Schering Corp. (Bloomfield, New Jersey, USA).
• Bis-(2,4-dinitrophenyl) oxalate was prepared according to the method of Rauhut et al, J. Amer. Chem. Soc. 89:6522(1967).
• Antibody reactive with sisomicin was raised in rabbits immunized with gentamicin coupled to bovine serum albumin [Nature New Biol. 239:214(1972)].
• silica gel (silica gel 60 brand, Merck, Darmstadt, West Germany) were used.
• the solvent was prepared by vigorously mixing equal volumes of methanol, chloroform and ammonium hydroxide overnight and collecting the resulting lower phase.
• Electrophoresis was conducted on 28 ⁇ 34 centimeter (cm) sheets of Whatman 3 MM brand paper (Reeve Angel, Clifton, New Jersey, USA) by the hanging strip method described by Williams et al, Science 121:829&830(1955).
• the buffer used was 0.01 molar (M) pyridine-acetate, pH 5.0. A potential of about 17 volts/cm was applied for 5 hours.
• Thin-layer chromatography resolved the commercial lissamine rhodamine B into 15 to 20 red components with the major constituent having an R f of 0.31.
• About 7 grams (g) of the crude mixture was stirred with 500 milliliters (ml) of boiling anhydrous ethanol and the resulting mixture was filtered while hot.
• the filtrate was passed into a 5 ⁇ 50 cm column of Sephadex LH-20 brand gel (Pharmacia Fine Chemicals, Piscataway, New Jersey, USA) in anhydrous ethanol.
• the column was washed with 6 liters of ethanol and 25 ml fractions were collected.
• the dry residue was dissolved in 3 to 4 ml dimethylacetamide and added to a solution containing 44 mg sisomicin (free base) (0.1 mmol) in 5 ml dimethylacetamide and 40 microliters ( ⁇ l) triethylamine (0.29 mmol).
• the reaction mixture stood at room temperature overnight and then the solvent was removed on a rotary evaporator.
• the residue was stirred with water and the insoluble material was separated by filtration.
• the water insoluble fraction dissolved in methanol and appeared to have a neutral charge when examined by electrophoresis.
• the water soluble material was composed of neutral and positively charged red components as well as unreacted sisomicin.
• Light-producing reactions were conducted in 7 ⁇ 70 mm test tubes mounted above a photomultiplier (type PM 270D from International Light, Newburyport, Mass., USA).
• a band pass interference filter (Ditric Optics, Marlboro, Mass., USA), with a 10 nm half band width was placed between the reaction tube and the photomultiplier. The wavelength for maximum transmission for the filter was 579 nm.
• the cover over the reaction tube had an opening covered by a rubber septum. A syringe needle was passed through the septum for injection of a solution of the oxalate ester into the reaction tube. Light production was recorded in terms of the units of the instrument reading.
• Binding reactions between antibody, the sisomicin-fluorescer conjugate and sisomicin were conducted in 0.1 M Tris-HCl buffer, pH 8.0, (400 ⁇ l final volume) at room temperature. The conjugate and sisomicin were combined and the antibody was added last. The binding reaction was allowed to proceed for 20 minutes. Then 150 ⁇ l of a solution of 500 g/liter Carbowax 6000 brand polyethyleneglycol (BDH Laboratories, Poole, England) in 0.1 M Tris-HCl, pH 8.0 was added and the protein precipitate which formed (including the "bound-species") was sedimented by centrifugation. Three hundred fifty microliter aliquots of the supernatants (the "free-species”) were transferred to clean 7 ⁇ 70 mm test tubes. Hydrogen peroxide was added and light measurements were conducted as outlined above.
• Variable levels of sisomicin and a fixed level of the sisomicin-fluorescer conjugate were allowed to react with a limited amount of antibody. Then the amount of unbound conjugate was measured with the light-producing reaction. Light production increased as the sisomicin level increased. At the highest level employed, the light production was 79% of that measured in the absence of the antibody.
• Sisomicin could be detected at levels as low as 1.3 ⁇ M.
• Table 2 demonstrate that the sisomicin-fluorescer conjugate is bound specifically by the antibody.

Landscapes

• Health & Medical Sciences (AREA)
• Immunology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Hematology (AREA)
• Urology & Nephrology (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Cell Biology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Priority Applications (5)

Applications Claiming Priority (1)

Publications (1)

Family

ID=21711472

Family Applications (1)

Country Status (5)

Cited By (49)

Families Citing this family (9)

Citations (16)

Family Cites Families (1)

• 1979
  • 1979-01-18 US US06/004,580 patent/US4238195A/en not_active Expired - Lifetime
  • 1979-12-10 CA CA341,540A patent/CA1133392A/en not_active Expired
  • 1979-12-27 DE DE2952498A patent/DE2952498C2/de not_active Expired
• 1980
  • 1980-01-02 GB GB8000086A patent/GB2044449B/en not_active Expired
  • 1980-01-17 JP JP314880A patent/JPS5596458A/ja active Granted

Patent Citations (19)

Non-Patent Citations (3)

Also Published As

Similar Documents